bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–05–04
forty-six papers selected by
Sofia Costa, Matterworks
  1. Development and Validation of a Rapid and Simple LC-MS/MS Method for Quantification of the Anti-Microtubule, Anti-Cancer Agent ABT-751 in Human and Mouse Plasma and Mouse Tissues.
  2. Determination of Efinaconazole in Plasma using Validated LC-MS/MS Technique.
  3. Integrating NMR and multi-LC-MS-based untargeted metabolomics for comprehensive analysis of blood serum samples.
  4. Isotope tracing-based metabolite identification for mass spectrometry metabolomics.
  5. Dual LC column characterization for mass spectrometry-based small molecule profiling of human plasma and serum.
  6. Method Validation for the Determination of Nicotine and Nicotine Metabolite Levels in Urine, Serum, and Saliva Samples.
  7. Determination of 59 Psychotropic Drugs, Including Antipsychotics, Antidepressants, Antiepileptics, Anti-Alcohol Abuse Drugs, Anti-ADHD Drugs, and Their Metabolites in Urine Using LC-MS/MS for Medication Compliance Monitoring.
  8. The current state in liquid chromatography-mass spectrometry methods for quantifying kynurenine pathway metabolites in biological samples: a systematic review.
  9. Automated Integration and Quality Assessment of Chromatographic Peaks in LC-MS-Based Metabolomics and Lipidomics Using TARDIS.
  10. Derivatization-Assisted Acoustic Ejection Mass Spectrometry for High-Throughput FAHFA Prescreening.
  11. Accurate Determination of Circulatory Lipids Using a Combination of HILIC-MRM and RPLC-PRM.
  12. Multistage fragmentation (MS3) detection enhances quantitative sensitivity and accuracy for LC-MS analysis of serum steroid hormones.
  13. Electromembrane Extraction in Suspect Screening of Polar Organic Xenobiotics and their Metabolites in Human Urine: A New Approach to Enhance Compound Annotation?
  14. Large-Scale Metabolite Imaging Gallery of Mouse Organ Tissues to Study Spatial Metabolism.
  15. Precision Chemotherapy: Optimizing Calibration for Rapid Determination of Blood Methotrexate by Tandem Mass Spectrometry ± Liquid Chromatography.
  16. Two-dimensional ion chromatography tandem-mass spectrometric (2D-IC-MS/MS) method for the analysis of phosphorus compounds in soil.
  17. COLMAR1d2d: Synergistic Combination of 1D with 2D NMR for Enhanced High-Throughput Identification and Quantification of Metabolites in Complex Mixtures.
  18. Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Method Development and Validation to Quantify Simultaneously Six Urinary DIALKYL Phosphate Metabolites of Organophosphorus Pesticides.
  19. Achiral LC-MS/MS and chiral SFC-MS methods for quantification of methoxphenidine and O-desmethyl-methoxphenidine metabolite in rat serum and brain.
  20. Effect of pH on stability and solid phase extraction of urinary free metadrenaline measurement by liquid chromatography tandem mass spectrometry.
  21. Rapid quantification of 21 antihypertensive and diuretic drugs in plasma by UPLC-MS/MS: Application to clinical and forensic cases.
  22. Rapid and Sensitive Detection of Quinones by In-Source Microdroplet Derivatization Coupled with Mass Spectrometry.
  23. Exploring Sample Storage Conditions for the Mass Spectrometric Analysis of Extracted Lipids from Latent Fingerprints.
  24. Analytical workflow for comprehensive blood metabolomics analysis by GC-MS. Application to children with ventilator associated pneumonia.
  25. A simplified one-pot derivatization/magnetic solid-phase extraction with integrated pH adjustment strategy coupled with liquid chromatography-fluorescence detection for fatty aldehyde analysis.
  26. N-Heterocyclic Carbenes: Novel Derivatization Reagents for LC-MS Analysis of Aliphatic Aldehydes.
  27. Analysis of neutral per- and polyfluoroalkyl substances (PFAS) by gas chromatography ‒ high resolution mass spectrometry (GCHRMS).
  28. A Laboratory Protocol for Routine Therapeutic Drug Monitoring of Beta-Lactams Antimicrobials in Horses and Dogs.
  29. Ultra-sensitive quantification of fipronil in zebrafish tissues via UPLC-MS3.
  30. Development of an Optimized Two-Step Solid-Phase Extraction Method for Urinary Nucleic Acid Adductomics.
  31. High-Resolution LC-MS Approach for In Vitro Metabolite Characterisation of Etonitazene in Camels for Enhanced Doping Control.
  32. Glucuronidation Metabolomic Fingerprinting to Map Host-Microbe Metabolism.
  33. Unspecified Degradation Impurities Identification and Characterization in Bilastine and Montelukast tablet formulations by using UPLC and LCMS/MS: Robustness by Design Expert and Green assessment.
  34. A Facile Workflow for Sebum Fatty Acids and Squalene Identification via Advanced Lipidomic Methodologies.
  35. Ultrahigh Flow Regulated Discharge Coupling with Targeted Mass Spectrometry Analysis: Real Time Capturing Biomolecules in Exhaled Breath.
  36. Volumetric absorptive microsampling to measure iohexol and creatinine concentrations for estimation of glomerular filtration rate in cats: aligning animal welfare with practical feasibility.
  37. Dual SPE-HPLC-MS/MS Platform for Cross-Class Antiparasitic Surveillance: Simultaneous Quantification of Oxyclozanide and Levamisole Hydrochloride in Ovine Tissues with Applications to Withdrawal Period Optimization.
  38. Towards the development of an alternative analysis method to determine lead in eyeliner cosmetics.
  39. Simultaneous determination of piperacillin, metronidazole, and tazobactam in plasma by UPLC-MS/MS and application to a pharmacokinetic study in pediatric liver transplant patients.
  40. Lab-made open-source controlled robotic workstation for sorbent-based dispersive microextraction of low-volume samples.
  41. Development and Validation of RP-HPLC Method for the Detection of N-Nitroso Meglumine in Pharmaceutical Products.
  42. Development of a Rapid LC-MS/MS Method and Its Application for the Pharmacokinetic Analysis of Pacritinib in Rats.
  43. Identification of novel oxidized phospholipids that activate platelet-activating factor receptor using HPLC fractionation and comprehensive LC-MS/MS analysis.
  44. Purification of testosterone and its metabolites in urine using two-dimensional high-performance liquid chromatography for 13C/12C ratios analysis by gas chromatography/combustion/isotope ratio mass spectrometry.
  45. MMEASE: enhanced analytical workflow for single-cell metabolomics.
  46. Development of CE-MS/MS method for unravelling nonsteroidal anti-inflammatory drugs as potential adulterants of Boswellia serrata extract.
  1. Biomed Chromatogr. 2025 Jun;39(6): e70099
    Development and Validation of a Rapid and Simple LC-MS/MS Method for Quantification of the Anti-Microtubule, Anti-Cancer Agent ABT-751 in Human and Mouse Plasma and Mouse Tissues.
    Aristeidis Lentzas, Anna Their, Jos H Beijnen, Mark C de Gooijer, Olaf van Tellingen.
      A rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantifying ABT-751, an anticancer agent targeting microtubules. Sample preparation involved protein precipitation using acetonitrile and formic acid (100:1, v/v), providing efficient ABT-751 extraction with minimal ion suppression. Buparlisib (BKM-120) served as the internal standard. Chromatographic separation was achieved on a Zorbax Extend C18 column, with gradient elution from 20 to 95% methanol in 0.1% (v/v) formic acid in water, and MS/MS detection was performed in positive ionization mode. This assay was validated for human plasma, mouse plasma, and various mouse tissues, including brain, liver, lung, and kidney homogenates. Calibrants were prepared in each respective blank biological matrix, except for mouse tumor tissue, and curves were fitted by quadratic regression from 5 to 10,000 nM. For mouse tumor tissue we used human plasma as surrogate matrix for calibrants. Precision and accuracy for intra-day and inter-day measurements were within acceptable limits across low, medium, and high concentrations for all matrices. Stability concerns with ABT-751 in mouse plasma and tissue homogenate samples that were stored for more than 8 months were identified and addressed. A pilot pharmacokinetic study in mice demonstrated the applicability of this validated LC-MS/MS method.
    Keywords:  ABT‐751 quantitation; LC‐MS/MS method validation; mouse plasma; protein precipitation; stability; tissue homogenates
    DOI:  https://doi.org/10.1002/bmc.70099
  2. Drug Metab Bioanal Lett. 2024 ;17(2): 76-87
    Determination of Efinaconazole in Plasma using Validated LC-MS/MS Technique.
    Govindarajan Srinivasan, Asharani Indira Viswambaran.
       BACKGROUND: Efinaconazole is a topical antifungal medication that is effective against fungal infections of the toenails. In addition, due to its application on the skin, minimal systemic absorption takes place on the epidermic layer, which leads to the availability of lower-level concentration in the bloodstream. Although several reported methods in the literature describe the quantification of Efinaconazole using conventional techniques like HPTLC and HPLC, these methods lack the necessary sensitivity and selectivity to be directly applied for quantification in biological samples.
    OBJECTIVE: Current research work aimed to develop a rapid, specific, selective, and sensitive method using plasma as one of the biological samples for quantification of Efinaconazole (EZ) in the presence of Fluconazole (FZ) as an internal standard by tandem mass spectrometry (LC-MS/MS).
    METHODS: Chromatographic separation was achieved with a Thermo Hypersil Gold (100 mm x 2.1 mm, 1.9 μm) UPLC column using a mobile phase composed of 20% formic acid-water (0.1%) and 80% methanol. Liquid-liquid extraction (LLE) was employed for sample preparation. Efinaconazole and the internal standard were detected using the heated electrospray ionization (HESI) technique in parallel reaction monitoring (PRM) mode.
    RESULTS: The developed method displayed a linearity range of 1 to 2000 pg/mL (0.001-2 ng/mL). Precision and accuracy for the lower limit of quantitation, low, mid, and high-quality control (QC) levels demonstrated a variance of less than 5% and an accuracy of 99 to 103%. Long-term stability was confirmed under various conditions, including storage in an auto-sampler, at room temperature, in a deep freezer, and after freeze-thaw cycles.
    CONCLUSION: The validated LC-MS/MS method has exceptional sensitivity, specificity, selectivity, rapid analysis, minimal requirement of sample quantity, wide dynamic range of concentration, robustness, and reproducibility, making it an indispensable tool, especially in fields of in vitro Permeation Testing (IVPT), in vitro Release Testing (IVRT), Pharmacokinetic, Toxicology, Clinical studies, and in drug development program for the quantification of Efinaconazole.
    Keywords:  LC-MS/MS; US-FDA.; efinaconazole; fluconazole; human plasma; validation
    DOI:  https://doi.org/10.2174/0118723128348675241129100845
  3. Anal Chim Acta. 2025 Jun 22. pii: S0003-2670(25)00373-3. [Epub ahead of print]1356 343979
    Integrating NMR and multi-LC-MS-based untargeted metabolomics for comprehensive analysis of blood serum samples.
    Tereza Kacerova, Elisabete Pires, John Walsby-Tickle, Fay Probert, James S O McCullagh.
       BACKGROUND: Mass spectrometry (MS) and nuclear magnetic resonance (NMR) have emerged as pivotal tools in biofluid metabolomics, facilitating investigation of disease mechanisms and biomarker discovery. Despite complementary capabilities, these techniques are rarely combined, although their integration is often beneficial. Typically, different sample preparation approaches are used, and compatibility challenges potentially arise due to the requirement for deuterated buffered solvents in NMR but not MS techniques. Additionally, MS-based approaches necessitate protein removal from samples whilst in NMR proteins can be potentially useful biomarkers. In this study, we developed a blood serum preparation protocol enabling sequential NMR and multi-LC-MS untargeted metabolomics analysis using a single serum aliquot in a research discovery setting.
    RESULTS: We analysed human serum samples using various untargeted NMR and multi-LC-MS platforms to assess the impact of deuterated solvents and buffers on detected compound-features. Employing multiple LC-MS profiling approaches, we observed no evidence of deuterium incorporation into metabolites following sample preparation with deuterated solvents. Furthermore, we demonstrated that buffers used in NMR were well tolerated by LC-MS. Protein removal, involving both solvent precipitation and molecular weight cut-off (MWCO) filtration, was identified as a primary factor influencing metabolite abundance. Our findings led to the development and validation of a serum sample preparation protocol enabling a combined NMR and multi-LC-MS analysis.
    SIGNIFICANCE: Using a single clinical serum aliquot for simultaneous untargeted profiling via NMR and multi-LC-MS represents a highly efficient alternative to current methods. This approach reduces sample volume requirements and substantially expands the potential for broader metabolome coverage. Our study offers comprehensive insights into the impact of sample preparation on complex metabolic biofluid profiles, highlighting the compatibility and complementarity of LC-MS and NMR in metabolomics research.
    Keywords:  Mass spectrometry; Multi-platform metabolomics; Nuclear magnetic resonance; Sample preparation; Untargeted metabolomics
    DOI:  https://doi.org/10.1016/j.aca.2025.343979
  4. bioRxiv. 2025 Apr 08. pii: 2025.04.07.647691. [Epub ahead of print]
    Isotope tracing-based metabolite identification for mass spectrometry metabolomics.
    Deniz Secilmis, Arjana Begzati, Nina Grankvist, Irena Roci, Jeramie Watrous, Amit R Majithia, Gordon I Smith, Samuel Klein, Mohit Jain, Roland Nilsson.
      Modern mass spectrometry-based metabolomics is a key technology for biomedicine, enabling discovery and quantification of a wide array of biomolecules critical for human physiology. Yet, only a fraction of human metabolites have been structurally determined, and the majority of features in typical metabolomics data remain unknown. To date, metabolite identification relies largely on comparing MS 2 fragmentation patterns against known standards, related compounds or predicted spectra. Here, we propose an orthogonal approach to identification of endogenous metabolites, based on mass isotopomer distributions (MIDs) measured in an isotope-labeled reference material. We introduce a computational measure of pairwise distance between metabolite MIDs that allows identifying novel metabolites by their similarity to previously known peaks. Using cell material labeled with 20 individual 13 C tracers, this method identified 62% of all unknown peaks, including previously never seen metabolites. Importantly, MID-based identification is highly complementary to MS 2 -based methods in that MIDs reflect the biochemical origin of metabolites, and therefore also yields insight into their synthesis pathways, while MS 2 spectra mainly reflect structural features. Accordingly, our method performed best for small molecules, while MS 2 -based identification was stronger on lipids and complex natural products. Among the metabolites discovered was trimethylglycyl-lysine, a novel amino acid derivative that is altered in human muscle tissue after intensive lifestyle treatment. MID-based annotation using isotope-labeled reference materials enables identification of novel endogenous metabolites, extending the reach of mass spectrometry-based metabolomics.
    DOI:  https://doi.org/10.1101/2025.04.07.647691
  5. Anal Chim Acta. 2025 Jun 22. pii: S0003-2670(25)00336-8. [Epub ahead of print]1356 343942
    Dual LC column characterization for mass spectrometry-based small molecule profiling of human plasma and serum.
    Žiga Tkalec, Štěpán Koudelka, Jana Klánová, Elliott J Price.
       BACKGROUND: Analyte annotation confidence in untargeted liquid chromatography mass-spectrometry (LC-MS) based chemical analysis can be enhanced by leveraging retention time information. For this, the chromatographic characteristics of the analytical system used should be well characterized. In this study, we measured 604 diverse chemical standards to characterize a dual LC setup consisting of pentabromobenzyl (PBr) and type-C silica hydride (SiH) columns operating in reversed-phase (RP) and aqueous normal-phase (ANP) mode, respectively.
    RESULTS: ANP and RP separations individually retained 40 % and 64 % of standards in cLogP range from -6.60 to 8.67 and -3.34 to 12.95, respectively. Using both columns, the coverage increased to 79 % of standards with cLogP range from -6.60 to 12.95 (median cLogP = 1.63). Retention selectivity follows the number of basic nitrogen atoms in the molecule on SiH column and polarity (cLogP) on PBr column. Column repeatability and reproducibility were tested in triplicate using a chemically diverse subset of 108 standards. Repeatability of retention times, peak widths and peak areas was 0.3 %, 14 %, 4 % for SiH column and 0.2 %, 12 %, 4 % for PBr column. Similarly, reproducibility was 15 %, 34 %, 30 % for SiH column and 9 %, 18 % and 34 % for PBr column. Predictive RT models were developed based on experimental RT data, achieving R2 values of 0.92 and 0.96, with mean absolute errors of 0.29 min and 0.27 min for SiH and PBr columns, respectively.
    SIGNIFICANCE: As proof of concept, 129 metabolites were annotated in pooled human serum and plasma by matching standard or predicted RT on one or both columns. The RT models and MS2 spectra of standards are openly available, facilitating uptake of this well-characterized chromatographic system to increase confidence in analyte annotation.
    Keywords:  Aqueous normal-phase; Chemicals; Exposome; Metabolomics; Non-targeted; Retention time prediction
    DOI:  https://doi.org/10.1016/j.aca.2025.343942
  6. Biomed Chromatogr. 2025 Jun;39(6): e70096
    Method Validation for the Determination of Nicotine and Nicotine Metabolite Levels in Urine, Serum, and Saliva Samples.
    Gülsüm Abuşoğlu, Duygu Eryavuz Onmaz, Ali Ünlü, Fatma Hümeyra Yerlikaya Aydemir, Sedat Abuşoğlu.
      Monitoring nicotine and its metabolites in bodily fluids is crucial for assessing tobacco exposure and related toxicity in clinical practice. Due to its high sensitivity for detecting low-level analytes, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to enhance a robust and accurate analytical procedure for quantifying nicotine and its derivatives in serum, urine, and saliva. Comparative analysis was conducted on samples collected from individuals with and without smoking habits. The LC-MS/MS method utilized an API 3200 triple quadrupole instrument paired with a Phenomenex Luna C18 column. Method sensitivity was demonstrated through evaluation of sensitivity parameters, including LOD and LLOQ for each analyte in all matrices. Inter-assay variability and bias values at the LLOQ level were kept below 20%. In serum, the LOD-LLOQ ranges were 0.02-0.05 μg/L for nicotine, 0.32-0.95 μg/L for cotinine, 0.07-0.25 μg/L for 3-OH cotinine, and 0.22-0.69 μg/L for norcotinine. In urine, the respective values were 0.03-0.10, 0.23-0.82, 0.06-0.14, and 0.20-0.55 μg/L, and in saliva, the values were 0.12-1.59, 0.22-2.34, 0.10-0.17, and 0.33-3.01 μg/L. The proposed method is efficient, specific, and adaptable to routine clinical workflows, providing a valuable tool for monitoring tobacco-related exposure.
    Keywords:  LC MS/MS; cotinine; method validation; nicotine
    DOI:  https://doi.org/10.1002/bmc.70096
  7. Biomed Chromatogr. 2025 Jun;39(6): e70074
    Determination of 59 Psychotropic Drugs, Including Antipsychotics, Antidepressants, Antiepileptics, Anti-Alcohol Abuse Drugs, Anti-ADHD Drugs, and Their Metabolites in Urine Using LC-MS/MS for Medication Compliance Monitoring.
    Jeong Eun Kim, Seon Yeong Kim, Jae Chul Cheong, Jin Young Kim.
      Increasing social concern regarding individuals with mental health disorders in the criminal justice system has underscored the need for effective strategies to reduce recidivism. Medication compliance monitoring is a critical approach that ensures adherence to prescribed treatments, facilitating the reintegration of these individuals into society. This study focuses on the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detecting 59 psychotropic drugs and their metabolites in urine, addressing a significant gap in existing monitoring techniques. Utilizing a dilute-and-shoot approach for urine sample preparation, we established a robust analytical method that demonstrated high sensitivity and precision, with limits of detection ranging from 0.07 to 1.5 ng/mL and correlation coefficients consistently above 0.997. The method was validated through various parameters, including selectivity, stability, and accuracy, ensuring reliable performance in forensic applications. Analysis of urine samples from 248 individuals on probation with mental health conditions confirmed the practicality of the method, identifying 53 psychotropic substances. The LC-MS/MS method was successfully applied for medication compliance monitoring of mentally disordered probationers. Therefore, this analytical method could provide clear evidence for preventing the recurrence of mentally disordered crimes.
    Keywords:  LC–MS/MS; medication compliance monitoring; psychotropic drug; urine
    DOI:  https://doi.org/10.1002/bmc.70074
  8. Crit Rev Clin Lab Sci. 2025 Apr 29. 1-17
    The current state in liquid chromatography-mass spectrometry methods for quantifying kynurenine pathway metabolites in biological samples: a systematic review.
    Md Munnaf Hossen, Bobbi Fleiss, Rosita Zakaria.
      Kynurenine pathway (KP) metabolites are implicated in various disorders, including Alzheimer's disease, schizophrenia, and adverse pregnancy outcomes. Simultaneous measurement of multiple KP metabolites offers valuable insight into the pathway's role in health and disease, would improve this relatively undeveloped field. This systematic review aim was to summarize the state of the art for measuring the eight key KP metabolites, using liquid chromatography-mass spectrometry (LC-MS), explicitly focusing on whether methods were validated using established guidelines with superior sensitivity and selectivity. We undertook a comprehensive review of the literature using the PRISMA guidelines. Our search uncovered 66 publications, and 39 qualified the defined key criteria. We summarized each publication's method development parameters, analytical design, and method performance specifications. We found notable variability in sample preparation techniques and analytical design across biological matrices, underscoring a lack of universally established and validated methods for KP metabolite quantification. We also identified significant gaps in the basic method evaluation. Our findings highlight that no single method has been evaluated for quantifying the eight key KP metabolites across three or more biological sample types, revealing a critical gap in the field. Our review emphasizes the need for robust analytical methods to quantify KP metabolites across multiple biological matrices, facilitating a better understanding of their roles in health and disease. Given the diversity of disorders involving the KP in the clinical testing lab, developing such methods will reduce diagnostic errors and advance KP metabolite research, supporting more precise, and personalized medical care.
    Keywords:  Bioanalytical methods; brain disorders; kynurenic acid; method evaluation; quinolinic acid
    DOI:  https://doi.org/10.1080/10408363.2025.2495160
  9. Anal Chem. 2025 Apr 28.
    Automated Integration and Quality Assessment of Chromatographic Peaks in LC-MS-Based Metabolomics and Lipidomics Using TARDIS.
    Pablo Vangeenderhuysen, Matthijs Vynck, Beata Pomian, Kimberly De Windt, Emile Callemeyn, Ellen De Paepe, Lindsey De Commer, Jeroen Raes, Tim Nawrot, Johannes Rainer, Lieselot Y Hemeryck, Lynn Vanhaecke.
      In recent years, liquid chromatography coupled to mass spectrometry (LC-MS) has emerged as the main technology to measure the whole of small molecules (the metabolome) in a diversity of matrices. Within the field of computational metabolomics, significant efforts have been made in the development of tools to (pre)process untargeted LC-MS data. However, tools that circumvent the time-consuming, manual preprocessing of targeted LC-MS data with vendor-specific software remain sparse. We therefore present TARDIS, an open-source R package for the analysis of targeted LC-MS metabolomics and lipidomics data. Both established (area under the curve, maximum intensity and points over the peak) and recently developed (custom signal-to-noise ratio and bell-curve similarity) quality metrics were included to offer increased efficiency of peak quality evaluation. The robustness of TARDIS' peak integration was demonstrated through a quantitative comparison to state-of-the-art vendor software. To this end, applicability at a large scale (n = 1786) was validated across three distinct biofluids (stool, saliva and urine) and two LC-MS instruments, using data from the FAME, ENVIRONAGE, and FGFP cohort studies. In conclusion, TARDIS offers a robust and scalable open-source solution for the targeted analysis of LC-MS metabolomics and lipidomics data. TARDIS and its source code are freely available at https://github.com/UGent-LIMET/TARDIS.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00567
  10. Anal Chem. 2025 Apr 30.
    Derivatization-Assisted Acoustic Ejection Mass Spectrometry for High-Throughput FAHFA Prescreening.
    Na An, Peng-Cheng Mei, Ao Li, Xiu-Feng Fu, Xin-Ze Wu, Han-Peng Jiang, Quan-Fei Zhu, Yu-Qi Feng.
      Fatty acid esters of hydroxy fatty acids (FAHFAs) are bioactive lipids with significant structural diversity and potential health benefits, playing crucial roles in metabolic regulation, inflammation, and insulin sensitivity. However, the low abundance of FAHFAs in biological samples (0.1% to 0.001% of free fatty acids) and the wide range of molecular polarities (ClogP values from 0.26 to 18.24) across short-chain to long-chain FAHFAs pose substantial challenges for high-throughput screening. Here, we developed a novel high-throughput prescreening strategy, termed DAEMS (derivatization-assisted acoustic ejection mass spectrometry), which integrates chemical derivatization with acoustic ejection mass spectrometry (AEMS). By leveraging the ultrafast analysis speed of AEMS (1-3 samples per second) and the sensitivity enhancement of N,N-dimethylethylenediamine (DMED) derivatization, DAEMS enables the screening of over 2800 potential FAHFA species in less than 2 h─significantly faster than the conventional LC-MRM MS-based screening approaches. The DAEMS strategy achieved 83% coverage and 79% accuracy in preliminary screening compared to the LC-MRM MS. Furthermore, we also revealed novel oxidized FAHFA species in edible fungi for the first time, suggesting potential biosynthesis involving oxylipins or oxidative modifications. This study demonstrates DAEMS as a promising tool for rapid FAHFA prescreening, and the discovery of oxidized FAHFAs provides new insights into FAHFA diversity and metabolism.
    DOI:  https://doi.org/10.1021/acs.analchem.5c01004
  11. Anal Chem. 2025 May 02.
    Accurate Determination of Circulatory Lipids Using a Combination of HILIC-MRM and RPLC-PRM.
    Kyeong-Seog Kim, Jae-Seung Lee, Seung Seok Han, Joo-Youn Cho.
      Circulatory lipids are important markers for characterizing disease phenotypes; however, accurately determining lipid species remains a significant challenge in lipidomic analysis. Here, we present a novel analytical workflow for accurate lipidome characterization in human plasma using mass spectrometry (MS) through the integration of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC). This workflow enables rapid screening of 1,966 lipid species across 18 lipid classes using HILIC-multiple reaction monitoring (MRM), which enables facile identification of lipid species by lipid class-based separations. In the NIST Standard Reference Material for Human Plasma (SRM 1950), 489 lipid species were identified using HILIC-MRM and subsequently analyzed with RPLC-parallel reaction monitoring (PRM) to resolve potential lipid isobars within the same lipid class. Notably, RPLC-PRM identified 70 additional lipidomic features in SRM 1950 that were not detectable with HILIC-MRM. Furthermore, a high correlation (Pearson correlation coefficient = 0.81) was observed regarding the concentrations of lipid species not carrying isobaric interferences in between HILIC-MRM and RPLC-PRM, indicating that the individual lipid concentrations measured by each platform can be integrated. The workflow was further applied to a cohort of 284 human plasma samples from chronic kidney disease (CKD) patients, successfully profiling lipidomic phenotypes across CKD subtypes. These findings demonstrate that combining HILIC-MRM and RPLC-PRM as complementary platforms enhances the accuracy and comprehensiveness of lipidomic analysis.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06409
  12. Talanta. 2025 Apr 23. pii: S0039-9140(25)00688-5. [Epub ahead of print]294 128198
    Multistage fragmentation (MS3) detection enhances quantitative sensitivity and accuracy for LC-MS analysis of serum steroid hormones.
    Keke Yi, Wei Wan, Ziyu Qu, Di Zhang, Zihong Ye, Xinhua Dai, Jie Xie, You Jiang, Xiang Fang.
      A determination method using a home-built quadrupole-linear ion trap tandem mass spectrometry (Q-LIT) multistage fragmentation function to accurate measure ultra-trace steroid hormones in human serum was established. The capability of ion trap for multistage fragmentation (MS3) overcomes the limitation of triple quadrupole systems restricted to MS2, enabling deeper structural characterization. Multistage fragmentation (MS3) enables the accurate detection of steroid hormones in serum samples without requiring extensive derivatization or time-consuming sample preparation and improves sensitivity by effectively reducing noise. Hydrocortisone, 4-androstenedione, 11-deoxycortisol, 21-deoxycortisol, and 17α-hydroxyprogesterone were quantified by MS3 transitions m/z 363.2 → 327.2→309.2, m/z 287.2 → 97.0→79.0, m/z 347.1 → 311.2→293.2, m/z 347.0 → 311.1→293.1, and m/z 331.2 → 313.3→295.2, respectively. Multistage fragmentation mass spectrometry effectively prevents matrix interference, and the quantitative results of MS3 are less affected by matrix effect than those of MS2. The established multistage fragmentation quantitative method showed good linearity (R2 ≥ 0.99), and the limits of detection and quantification of 5 steroid hormones were lower than 0.06 and 0.20 ng/mL, respectively. The recovery was 86.56 %-123.14 %, with an RSD of ≤13.57 %. A total of 22 real serum samples were accurately and sensitively measured using the Q-LIT multistage fragmentation method. Therefore, Q-LIT based multistage fragmentation has great potential for applications, such as detecting steroid hormones with severe matrix effects and distinguishing among related isomers in complex biological samples.
    Keywords:  LC–MS; Linear ion trap; Multistage fragmentation; Serum; Steroid hormone
    DOI:  https://doi.org/10.1016/j.talanta.2025.128198
  13. Anal Chem. 2025 Apr 29.
    Electromembrane Extraction in Suspect Screening of Polar Organic Xenobiotics and their Metabolites in Human Urine: A New Approach to Enhance Compound Annotation?
    Mikel Musatadi, Ali Sahragard, Eneritz Anakabe, Nestor Etxebarria, Maitane Olivares, Olatz Zuloaga, Manuel Miró.
      Suspect and nontarget screening (SNTS) methodologies using human urine are invaluable strategies for understanding the human exposome. However, very polar organic compounds are often overlooked in those methods due to challenges in sample preparation and chromatographic analysis. Although "dilute-and-shoot" (DS) followed by mixed-mode liquid chromatography-high-resolution mass spectrometry (MMLC-HRMS) might be deemed suitable, complementary strategies are needed to enhance SNTS and expand compound identification. In this context, the potential of nonsupported microelectromembrane extraction (μ-EME) is thoroughly studied as a supplement to DS-MMLC-HRMS. It was demonstrated that μ-EME-MMLC-HRMS enables the refinement of suspect screening results from a 24 h pooled human urine sample. The selective extraction capability of μ-EME for charged analytes, compared to DS, allowed the identification of 24 false positives and 4 false negatives. The confidence level of 6 suspects was also enhanced through μ-EME interpretation. Moreover, nine suspects were identified exclusively in μ-EME experiments due to the urine cleanup provided by that technique. Notably, suspects containing carboxylic acid groups (phase II metabolites) and amines were particularly well-annotated by μ-EME employing selective extraction conditions for acids and bases, respectively. Thus, μ-EME proves to be a confirmatory dimension in MMLC-based SNTS approaches.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06118
  14. J Proteome Res. 2025 Apr 28.
    Large-Scale Metabolite Imaging Gallery of Mouse Organ Tissues to Study Spatial Metabolism.
    Karl W Smith, Antonia Fecke, Siva Swapna Kasarla, Prasad Phapale.
      Mass spectrometry imaging (MSI) has revolutionized the study of tissue metabolism by enabling the visualization of small molecule metabolites (SMMs) with high spatial resolution. However, comprehensive SMM imaging databases for different organ tissues are lacking, hindering our understanding of spatial organ metabolism. To address this resource gap, we present a large-scale SMM imaging gallery for mouse brain, kidney, and liver, capturing SMMs spanning eight chemical super classes and encompassing over 40 metabolic pathways. Manual curation and display of these imaging data sets unveil spatial patterns of metabolites that are less documented in the reported organs. Specifically, we identify 65 SMMs in brain coronal sections and 71 in sagittal tissue sections, including spatial patterns for neurotransmitters. Furthermore, we map 98 SMMs in kidneys and 66 SMMs in liver, providing insights into their amino acid and glutathione metabolism. Our insightful SMM imaging gallery serves as a critical resource for the spatial metabolism research community, filling a significant resource gap. This resource is freely available for download and can be accessed through the BioImage Archive and METASPACE repositories, providing high-quality annotated images for potential future computational models and advancing our understanding of tissue metabolism at the spatial level.
    Keywords:  MALDI-MSI; imaging database; mass spectrometry imaging; metabolite imaging; organ metabolism; small molecules; spatial metabolomics; tissue metabolism
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00594
  15. Rapid Commun Mass Spectrom. 2025 Jul 30. 39(14): e10053
    Precision Chemotherapy: Optimizing Calibration for Rapid Determination of Blood Methotrexate by Tandem Mass Spectrometry ± Liquid Chromatography.
    Dennis J Dietzen, Connor J Blair.
       BACKGROUND: Rapid therapeutic monitoring of high-dose methotrexate (MTX) chemotherapy is essential to avoid toxicity. MTX concentrations are commonly monitored using immunoassays that are limited by narrow dynamic range and metabolite cross reactivity. Mass spectrometry may improve the molecular specificity of MTX analysis but is limited by slow throughput and extensive calibration. In this study, we examined the consequences of eliminating LC from MS/MS and foregoing external calibration to enable rapid determination of MTX.
    METHODS: MTX (m/z 455 → 308), was assessed using UPLC-MS/MS or flow-injection MS/MS using a six-point external calibration scheme with a single deuterated internal standard, 4-point internal calibration using 13C5, 13C6, 13C11, and 13C14-MTX, or by single point calibration with the single deuterated internal standard. Accuracy, precision, and resistance to hemolysis, icterus, and lipemia were compared with immunoassay.
    RESULTS: Across all six injection/calibration schemes, imprecision ranged from 2.5% to 10% (CV) from 0.05-10 μM. Regardless of calibration scheme, MS/MS ± LC was more resistant to interference from hemolysis and bilirubin than immunoassay. MS/MS ± LC determination of MTX was compared with immunoassay in 81-100 plasma specimens with MTX concentrations ranging from 0.05-81 μM. Intercept estimates all included zero with 95% confidence. Estimates of slopes versus immunoassay for each of the six analytic approaches ranged from 0.88 to 1.09.
    CONCLUSIONS: Eliminating LC and external calibration enabled rapid, precise, and accurate determination of MTX concentrations in plasma. Minimalist but robust approaches such as these may enable the use of MS for routine MTX determination in time-sensitive clinical circumstances.
    Keywords:  calibration; chemotherapy; chromatography; flow‐injection; methotrexate
    DOI:  https://doi.org/10.1002/rcm.10053
  16. J Chromatogr A. 2024 Aug 19. pii: S0021-9673(24)00661-7. [Epub ahead of print]1752 465287
    Two-dimensional ion chromatography tandem-mass spectrometric (2D-IC-MS/MS) method for the analysis of phosphorus compounds in soil.
    George Gachumi, Aimée Schryer, Steven D Siciliano.
      Investigations into soil organic phosphorus (Po) dynamics are instrumental in understanding the transformations and processes responsible for ecosystem productivity. However, quantitative analysis of Po in a soil environment is extremely challenging due to low target analyte concentrations and matrix interferences with chromatography and analysis. Consequently, a two-dimensional ion chromatography-tandem mass spectrometric (2D-IC-MS/MS) method was developed to estimate soil Po concentrations. The first dimension diverted early eluting anions to waste while preconcentrating P compounds in a trap column, followed by chromatographic separation and detection in the second dimension. Detection was done using a mass spectrometer, and quantification was performed using the multiple reaction monitoring scan (MRM) method. The linear range of the studied P compounds, mostly nucleotides, was 0.05-50 ng/mL. Most P compounds were detected and quantified in calcareous subsoil samples in the concentration ranges 0.70-51.78 ng/g. The developed method achieved chromatographic separation that allowed unambiguous identification of isobars/isomers and isotopologues contributing to interferences in MS detection. However, improvements to the extraction method and post-clean-up procedures are required due to the complexity of soil extract composition, extreme matrix effect and/or loss of analyte during preconcentration. The method is ideal for simultaneously analyzing P compounds from environmental samples to elucidate key components of the soil P dynamics.
    Keywords:  Ion chromatography; Mass spectrometry; Phosphate; Phosphorus compounds; Two-dimension chromatography
    DOI:  https://doi.org/10.1016/j.chroma.2024.465287
  17. Anal Chem. 2025 May 01.
    COLMAR1d2d: Synergistic Combination of 1D with 2D NMR for Enhanced High-Throughput Identification and Quantification of Metabolites in Complex Mixtures.
    R Cabrera Allpas, D-W Li, M Choo, K Lee, L Bruschweiler-Li, R Brüschweiler.
      A major challenge in 1D 1H NMR-based metabolomics studies is the occurrence of slight shifts of the resonances of mixture compounds compared to the reference spectra in the metabolomics spectral databases due to variations in buffer conditions, temperature, and matrix effects. This hampers both the automated spectral deconvolution and metabolite quantification of crowded regions in 1D 1H NMR spectra of complex mixtures whose analysis is particularly susceptible to such effects. 2D NMR-based metabolomics, on the other hand, is substantially more robust but also much more demanding in terms of NMR spectrometer time. Here we introduce an approach, termed COLMAR1d2d, which uses selected 2D 1H-13C HSQC and 1H-1H TOCSY NMR spectra of a subset of samples along with 1D 1H NMR spectra of all samples to overcome this bottleneck. It relies on our 2D NMR-based platform COLMARm using 2D 1H-13C HSQC and 2D 1H-1H TOCSY spectra measured for a representative subset of samples to unambiguously and comprehensively determine the metabolite composition and the exact peak positions of the identified compounds under the sample conditions present. This information is then used to update the spectral database for the automated analysis of a potentially large cohort of 1D 1H NMR spectra using the COLMAR1d platform. It is demonstrated how this synergistic combination of 1D with selected 2D NMR spectra allows the analysis of a significantly larger number of metabolites than would be possible with 1D NMR alone. Moreover, COLMAR1d2d also improves quantitation, as is demonstrated for samples from mouse urine and Pseudomonas aeruginosa biofilm.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00957
  18. J Mass Spectrom. 2025 May;60(5): e5128
    Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Method Development and Validation to Quantify Simultaneously Six Urinary DIALKYL Phosphate Metabolites of Organophosphorus Pesticides.
    Ngo Kien Đuc, Truong Nhat Khanh, Phan Nguyen Truong Thang, Tran Viet Hung, Pham Diem Thu, Le Thi Bich Chi, Vo Thi Kim Khuyen, Nguyen Duc Tuan.
      The exposure to chemical pesticides is one of the world's concerns, especially in Vietnam, which is becoming a key player in global agriculture. The chronic long-term exposure to pesticides, especially organophosphorus groups poses increased health risks such as cancer and related diseases due to their hazardous metabolites. The characterization of urinary pesticides is essential to understand the pesticide exposure patterns. Therefore, this research aims to develop a liquid chromatography-tandem mass spectrometry procedure for the quantification of six dialkyl phosphate metabolites of organophosphorus pesticides based on simulated human urine containing dialkyl phosphates and urine samples of organophosphorus pesticides-exposed farmers in An Giang province of Vietnam. As a result, a highly sensitive procedure with a negative ion atmospheric pressure chemical ionization source, fosfomycin as internal standard and multireaction monitoring, was successfully validated in compliance with international guidelines for simultaneous quantitative determination of six dialkyl phosphates in human urine samples. Molecular and fragmented ions for quantification were consistent with the standard spectrum. The linear ranges of DMP, DEP, DMTP, DETP, DMDTP, and DEDTP were 5.29-1000.58, 5.10-1000.19, 5.10-1000.20, 5.06-1000.11, 5.06-1000.11, 5.30-1000.60, and 5.06-1000.12 ng/mL, respectively. The validation results showed that the selectivity, intraday and interday precision and accuracy, matrix effect, carry over, dilution, and stability of all the analytes were in the acceptable range. In total, 383 spot urine samples from people working with pesticides were satisfactorily analyzed by the proposed procedure. Over 80% of farmers were detected with at least one organophosphate metabolite, especially DEDTP with high concentrations, up to 5015.0 ng/mL, which alerts the high likelihood of pesticide exposure in the community of rural areas in Vietnam.
    Keywords:  An Giang province; LC–MS/MS; dialkyl phosphates; human urine; organophosphate pesticides
    DOI:  https://doi.org/10.1002/jms.5128
  19. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Apr 24. pii: S1570-0232(25)00167-9. [Epub ahead of print]1259 124613
    Achiral LC-MS/MS and chiral SFC-MS methods for quantification of methoxphenidine and O-desmethyl-methoxphenidine metabolite in rat serum and brain.
    Natalie Paškanová, Magdaléna Vágnerová, Bronislav Jurásek, Kristýna Mazochová, Lucie Olejníková-Ladislavová, Tomáš Páleníček, Michal Kohout, David Sýkora, Martin Kuchař.
      Methoxphenidine (MXP), a dissociative anaesthetic derivative, has garnered the attention of toxicologists for their increasing abuse and associated toxicity. Despite that there is limited forensic and clinical toxicology data on MXP, especially regarding its metabolism and enantiomers. To fill this gap, we developed, validated and applied achiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chiral supercritical fluid chromatography-MS (SFC-MS) methods to quantify MXP and its primary metabolite, O-desmethyl-methoxphenidine (dmMXP), in rat serum and brain samples collected after a single subcutaneous dosing of racemic MXP. Serum samples were extracted by protein precipitation with 0.1 % formic acid in acetonitrile, and salting-out-assisted liquid-liquid extraction was utilised for brain extraction. The samples were analysed by a reversed-phase LC-MS/MS method equipped with a Poroshell 120 phenyl-hexyl column, and the enantioselective SFC-MS method was equipped with an Alcyon Amylose-SA column. Both methods were fully validated according to the European Medicines Agency guidelines. The LC-MS/MS method had a total run time of 4.8 min with a linear response up to 400 ng/mL in serum and 2400 ng/g in brain, and the limits of quantification were 1.00 ng/mL and 6.00 ng/g for MXP and 1.00 ng/mL and 1.50 ng/g for dmMXP. The enantioselective SFC-MS method had a total run time of 15 min, showing linear ranges up to 1000 ng/mL for individual enantiomers in serum and 7200 ng/g in brain. The limits of quantification of (R,S)-MXP were 12.5 ng/mL and 30.0 ng/g, while those of (R,S)-dmMXP were 25.0 ng/mL and 60.0 ng/g. The achiral LC-MS/MS method enabled quantification of racemic MXP and dmMXP, while the chiral SFC-MS method was used only for MXP enantiomers, as its higher lower limit of quantification did not allow for enantioselective quantification of dmMXP. Serum MXP peaked at 1600 ng/mL (0.5 h) and decreased to 5.87 ng/mL at 24 h, while dmMXP peaked at 11.8 ng/mL (1 h) and was below the limit of quantification at 24 h. In brain, MXP peaked at 13200 ng/g (0.5 h) and decreased to 36.1 ng/g (24 h), while dmMXP reached 67.1 ng/g (1 h) and decreased to 1.63 ng/g (24 h). Moreover, it was found that the (S)-MXP concentrations in the brain appeared to be higher than the (R)-enantiomer concentrations. The validated methods allow for the generation of pharmacokinetic curves for MXP within a behavioural study and provide a valuable tool for forensic and clinical toxicology for the study of the dissociative anaesthetic MXP and its metabolite in rat serum and brain.
    Keywords:  Chiral SFC-MS; Dissociative anaesthetics; Enantiomers; LC-MS/MS; Methoxphenidine; Quantification; Rat serum and brain
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124613
  20. Ann Clin Biochem. 2025 Apr 29. 45632251342098
    Effect of pH on stability and solid phase extraction of urinary free metadrenaline measurement by liquid chromatography tandem mass spectrometry.
    Lindsay McDonald, Craig Livie, Karen Smith, Susan Johnston.
       BACKGROUND: Measurement of urine free metadrenalines offers potential diagnostic and practical advantages over urinary fractionated metadrenalines in detection of phaeochromocytoma and paraganglioma, including sample collection without acid preservative. Here we evaluate stability with and without sample acidification as well as pH implications for analysis by solid-phase extraction (SPE) and liquid chromatography tandem mass spectrometry.
    METHODS: Spot urine samples were adjusted to pH 3 or unacidified on day of collection and stored at room temperature, 4 ºC or -20 ºC for up to 28 days to assess changes in free metadrenaline concentrations over time. Extraction of unacidified vs acidified urine was examined by comparing peak areas and measuring concentrations present in sample eluents according to two SPE methodologies.
    RESULTS: Free metadrenalines remained stable in urine with or without acidification for up to 28 days, with mean reduction in concentrations of <10 % for all storage conditions. Measured concentrations progressively increased without acidification at room temperature at low concentrations but remained constant when spiked with pathological concentrations. Peak areas were up to 97-fold lower in acidified than unacidified samples when extracted using weak cation exchange (WCX). On average 64 % of analyte eluted in the flowthrough in acidified samples relative to 1.5 % without acidification. By contrast, over 99 % was retained in the extract using polar extraction at either pH.
    CONCLUSION: Urine free metadrenalines remain stable at room temperature for up to 28 days and are more efficiently extracted without use of acid preservative if using WCX methodology.
    Keywords:  Clinical studies; Laboratory methods; Mass spectrometry; Tumour markers
    DOI:  https://doi.org/10.1177/00045632251342098
  21. J Pharm Biomed Anal. 2025 Apr 19. pii: S0731-7085(25)00251-1. [Epub ahead of print]263 116910
    Rapid quantification of 21 antihypertensive and diuretic drugs in plasma by UPLC-MS/MS: Application to clinical and forensic cases.
    Coralie Boudin, Amandine Faure, Alexandre Behouche, Olivier Ormezzano, Hélène Eysseric-Guérin, Françoise Stanke-Labesque, François Paysant, Virginie Scolan, Théo Willeman.
      Systemic arterial hypertension, affecting more than 1 billion people worldwide, necessitates widespread use of antihypertensive and diuretic medications. However, the potential toxicity related to exposure of these medications is not always fully understood, potentially leading to underestimates of deaths related to cardiovascular drugs. Additionally, the growing interest in monitoring adherence to antihypertensive medications necessitates the development of specific analytical methods suitable for both clinical and forensic applications. In this study, we developed a novel, high-throughput quantitative method for the simultaneous analysis of 21 antihypertensive and diuretic drugs mainly in human plasma using liquid chromatography with tandem mass spectrometry. This method has several advantages, including minimal sample volume requirement, a one-step sample preparation using an Ostro® plate, and a chromatographic run time of 7 min. The method was successfully validated on 11 criteria following the European Medicines Agency's guidances. The method was successfully applied to authentic samples from 62 clinical cases and 76 post-mortem cases, with two cases of severe intoxications more precisely described. The first case describes an attempted suicide by candesartan (2558 ng/mL in plasma) combined with celiprolol (18 ng/mL) and amlodipine (161 ng/mL). The second case is a diuretic-contaminated dietary supplement poisoning with plasma concentrations of 40 ng/mL for furosemide and 36 ng/mL for hydrochlorothiazide. The authors present a simple, fast, and sensitive quantification method for the analysis of 21 antihypertensive and diuretic drugs, with concentration values reported in both living subjects and post-mortem cases to aid in the often-challenging interpretation of cardiotropic drug concentrations.
    Keywords:  Antihypertensive; Clinical toxicology; Diuretics; Forensic toxicology; Hypertension; Liquid chromatography tandem mass spectrometry; Plasma concentration
    DOI:  https://doi.org/10.1016/j.jpba.2025.116910
  22. Anal Chem. 2025 May 02.
    Rapid and Sensitive Detection of Quinones by In-Source Microdroplet Derivatization Coupled with Mass Spectrometry.
    Lulu Feng, Siyuan Tan, Simin Cheng, Hongru Feng, Zehu Xie, Xiang Fang, Xiaoyun Gong, Yuanjiang Pan, Xinhua Dai.
      Quinones are highly reactive oxidants that pose risks of cytotoxicity and genotoxicity to the human body. Sensitive analysis of quinone pollutants by mass spectrometry remains challenging due to the very limited ionization efficiency of quinones and the long pretreatment time. Here, we developed a rapid and highly sensitive in-source microdroplet derivatization strategy for the determination of quinones in complex matrices. Triphenylphosphine was used as a novel "tag" to react with quinone for online derivatization of quinones via the microdroplet-driving conjugate addition reaction. The formation of phosphonium ions significantly increased the ionization efficiency of the quinones. The sensitivity was improved by 2-3 orders of magnitude as compared with other online derivatization methods. Optimization results showed that the nebulization gas pressure, capillary temperature, sample solvent concentration, and reagent concentration played important roles in the derivatization and ionization of the quinones. The developed method featured good linearity (R2 ≥ 0.996), high sensitivity (LODs at the nmol/L level), and qualified precision (RSDs ≤ 7.3%) for four typical quinones. The method was successfully applied to the determination of quinones in complex matrices including human serum and urine. Taking into consideration the fact that many derivatization procedures are completed in bulk solutions, the proposed in-source microdroplet derivatization method offers us an effective way for fast screening of derivatization reagents. The present work expands the utility of microdroplet chemistry as well as chemical derivatization in the analysis of trace compounds, which might have potential applications in the fields of environmental sciences and clinic analysis.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06780
  23. Biomolecules. 2025 Mar 25. pii: 477. [Epub ahead of print]15(4):
    Exploring Sample Storage Conditions for the Mass Spectrometric Analysis of Extracted Lipids from Latent Fingerprints.
    Aleesa E Chua, Eden P Go, Heather Desaire.
      In large-scale studies, uncontrolled systematic variability introduced during sample preparation, processing, and storage can interfere with the detection of subtle biological signals. This study evaluates storage conditions, including two sample preparation methods and storage durations, to minimize systematic variability in the analysis of extracted lipids from latent fingerprints. In the traditional approach, samples are prepared immediately, stored as lipid extracts, and processed in multiple batches. In an alternative method, samples are stored directly on the deposition foil, and preparation is delayed until all can be processed in a single batch. Storage duration is evaluated to determine if shorter storage with analysis in multiple batches is more effective than longer storage with analysis in a single batch. Our findings demonstrate that storage of latent fingerprint samples on the deposition foil is a viable option, with minimal degradation of key features even after eight months of storage. While some differences in lipid profiles were observed across storage conditions, these differences were minor and would likely have little impact in larger studies where biological variability is greater. These insights offer practical guidance for implementing latent fingerprint sampling in large-scale studies by identifying optimal conditions that preserve sample quality and streamline workflows.
    Keywords:  data analysis; lipids; mass spectrometry; sample processing; storage; variability
    DOI:  https://doi.org/10.3390/biom15040477
  24. J Chromatogr A. 2025 Mar 31. pii: S0021-9673(25)00272-9. [Epub ahead of print]1753 465924
    Analytical workflow for comprehensive blood metabolomics analysis by GC-MS. Application to children with ventilator associated pneumonia.
    M Ioannidis, T Mouskeftara, E Iosifidis, M Simitsopoulou, E Roilides, H Gika, María Fernanda Rey-Stolle, C Virgiliou.
      Metabolomics is a widely used approach for analyzing a vast array of low molecular weight compounds such as amino acids, organic acids, vitamins, biogenic amines and carbohydrates in biological samples, with the aim of investigating biomarkers in personalized medicine studies. Advancements in gas chromatography- mass spectrometry (GC-MS) instrumentation, along with the availability of commercial and public spectral libraries, have highlighted the relevance of GC-MS analysis as a valuable tool for metabolomics applications. Stability assessment in derivatisation and GC-MS analysis is a crucial yet often overlooked aspect of metabolomics studies. In this study, an untargeted GC-MS method workflow for large scale metabolomics studies is presented after assessment and optimization of whole blood sample's stability. The method consists of a common two-step derivatisation procedure including methoximation using methoxyamine hydrochloride, followed by silylation with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). To ensure the stability of the studied metabolites, extensive stability experiments were performed. The stability of the derivatives was evaluated over 24 h in the autosampler at room temperature, as well as after storage for 24 and 48 h at -20 °C for both derivatized and dried extracts. While derivatised samples remained stable for 24-48 h in the freezer, dried extracts exhibited variability after 48 h. Findings support the storage of derivatised samples over dried extracts, ensuring greater stability. To increase condidence in metabolite identification data from the analysis of 120 standard compounds were utilized. The developed method was applied to analyze blood samples from 32 children with ventilator-associated pneumonia (VAP), collected at four different time points during ICU hospitalization. This analysis led to the identification of 43 metabolites. The results of multivariate and univariate statistical analyses demonstrated several statistically significant metabolites, including aspartic acid, alanine, and pyroglutamic acid, which showed a strong correlation with the disease's manifestation and may potentially serve as biomarkers in the diagnosis of ventilator-associated pneumonia VAP at the stage of clinical suspicion.
    Keywords:  Blood; Derivatisation; GC-MS; Metabolomics; Stability; Ventilator-associated pneumonia
    DOI:  https://doi.org/10.1016/j.chroma.2025.465924
  25. J Chromatogr A. 2025 Apr 24. pii: S0021-9673(25)00336-X. [Epub ahead of print]1753 465988
    A simplified one-pot derivatization/magnetic solid-phase extraction with integrated pH adjustment strategy coupled with liquid chromatography-fluorescence detection for fatty aldehyde analysis.
    Nian Shi, Yuyang Wang, Mengyuan Lv, Xudong Xia, Zihan Li, Di Chen, Ranran Duan, Jinghua Zhang.
      A novel one-pot derivatization/magnetic solid-phase extraction with integrated pH adjustment (OPD/MSPE-pH) strategy was developed for the efficient and selective quantification of fatty aldehydes. This method seamlessly integrates pH regulation, derivatization, and magnetic separation in a single incubation step, where the sample solution, derivatization reagent, and magnetic composite (Fe₃O₄/MWCNTs-OH/CA) are processed together, significantly simplifying sample preparation. The magnetic composite, synthesized from Fe₃O₄ nanoparticles, hydroxylated multi-walled carbon nanotubes (MWCNTs-OH), and citric acid (CA), functions both as an acidic pH regulator and an efficient adsorbent for aldehyde-fluorenylmethyl carbazate (FMC) derivatives. Compared to conventional approaches, the OPD/MSPE-pH method exhibited superior operation efficiency, achieving low limits of detection (1.1-2.4 nM) and high recoveries (91.7-106.1 %). The optimized procedure was successfully applied to plasma and spiked plasma samples, demonstrating its effectiveness in complex biological matrices. This streamlined technique eliminates the need for additional pH-adjusting reagents and centrifugation, offering a practical, rapid, and highly efficient solution for fatty aldehyde analysis. Overall, the proposed OPD/MSPE-pH method provides a robust, sensitive, and reliable analytical platform for fatty aldehyde analysis, with potential applications in broader fields with suitable modifications.
    Keywords:  Chemical derivatization; Fatty aldehydes; Liquid chromatography-fluorescence detection; Magnetic solid-phase extraction; One-pot sample preparation; PH adjustment; Plasma
    DOI:  https://doi.org/10.1016/j.chroma.2025.465988
  26. Anal Chem. 2025 May 02.
    N-Heterocyclic Carbenes: Novel Derivatization Reagents for LC-MS Analysis of Aliphatic Aldehydes.
    Yan-Zhen Wang, Hua-Ming Xiao, Pei-Rong Bai, Xiang Liu, Xin Liu, Quan-Fei Zhu, Yu-Qi Feng.
      N-heterocyclic carbenes (NHCs) are versatile catalysts for organic reactions, characterized by their unique electron-donating properties and high activity. This study introduces NHCs as innovative derivatization reagents for liquid chromatography-mass spectrometry (LC-MS) analysis of aliphatic aldehydes. Five distinct NHC reagents were evaluated, and 2-mesityl-2,5,6,7-tetrahydropyrrolo[2,1-c][1,2,4]triazol-4-ium chloride (MTPTC) was identified as the most promising candidate due to its rapid reaction kinetics, high selectivity, and excellent product stability. The MTPTC-based derivatization reaction effectively addressed the issue of stereoisomeric products, resulting in well-resolved single peaks in the LC separation. Additionally, the derivatized products exhibited high stability, facilitating accurate and reliable quantitative analysis. Using MTPTC as the derivatization reagent, an LC-MS quantitative analysis strategy was developed for the determination of eight aliphatic aldehydes in human sera. The method demonstrated a broad linear range, low limits of detection and quantification, and satisfactory reproducibility and accuracy. The applicability of this method was further validated through the quantification of aliphatic aldehydes in the serum of sepsis patients. This work extends NHCs' utility to analytical chemistry and introduces a novel derivatization reagent for the analysis of carbonyl compounds by LC-MS.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06809
  27. J Chromatogr A. 2025 Apr 25. pii: S0021-9673(25)00337-1. [Epub ahead of print]1753 465989
    Analysis of neutral per- and polyfluoroalkyl substances (PFAS) by gas chromatography ‒ high resolution mass spectrometry (GCHRMS).
    Yelena Sapozhnikova, Kevin Stroski.
      Most of the studies on per- and polyfluoroalkyl substances (PFAS) to date encompass water soluble and ionic PFAS analyzed by liquid chromatography ̶ mass spectrometry, yet analytical methods and information on the occurrence of neutral PFAS are lacking. To this aim, we developed a new method for analysis of forty neutral PFAS using gas chromatography (GC) ̶ Orbitrap mass spectrometry with electron ionization (EI). Analytes were comprised of 29 fluorotelomer alcohols, 6 fluorotelomer acrylates and methacrylates, 3 perfluoroalkane sulfonamides and 2 perfluoroalkane sulfonamido alcohols. Gas chromatographic separation was developed on two GC phases: a standard non-polar (5 % diphenyl and 95 % dimethyl polysiloxane) and a mid-polar (6 % cyanopropylphenyl, 94 % dimethylpolysiloxane). A custom-made high-resolution mass spectral (HRMS) library was developed and used to evaluate PFAS accuracy of identification. Overall, 85 % of PFAS were correctly identified. A quantitative method was developed and evaluated for sensitivity, linearity, reproducibility, and ion interferences. Method sensitivity varied for different PFAS from 1 to 50 ppb based on the lowest calibrated levels. The developed method was utilized for analysis of PFAS in paper-based food contact materials after developing and evaluating the extraction protocol. Method applicability was demonstrated by analyzing paper-based food packaging samples, where 6:2 fluorotelomer alcohol was detected and measured at levels up to 351 ng/g. The developed GCHRMS method can be utilized for identification and measurement of neutral PFAS in various matrices, including food, dust, food contact materials, textiles and others.
    Keywords:  Acrylates; Analysis; Fluorotelomer alcohols; Gas chromatography; High resolution mass spectrometry; Sulfonamides
    DOI:  https://doi.org/10.1016/j.chroma.2025.465989
  28. Antibiotics (Basel). 2025 Apr 09. pii: 390. [Epub ahead of print]14(4):
    A Laboratory Protocol for Routine Therapeutic Drug Monitoring of Beta-Lactams Antimicrobials in Horses and Dogs.
    Anisa Bardhi, Aliai Lanci, Aurora Mannini, Carolina Castagnetti, Andrea Barbarossa.
      Background: Although antibiotic resistance is a well-known issue in veterinary medicine, studies proposing real-time therapeutic monitoring (TDM) are lacking. The objective of the present study was to develop a simple and rapid protocol for the real-time therapeutic monitoring of antibiotics in horses and dogs. Methods: A reliable TDM protocol should encompass guidelines for the definition of plasma/serum collection time points, sample management by the clinical staff, transportation to the laboratory, and the availability of robust and swift analytical technologies. Ampicillin and sulbactam were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the plasma or serum of animals treated with ampicillin alone or combined with sulbactam. Results: The method was successfully applied to samples collected from animals hospitalized in our veterinary hospital and proved helpful in understanding the pharmacokinetics of this antibiotic in critically ill patients. Conclusions: Combined with minimum inhibitory concentration (MIC) data, this approach enables PK/PD evaluations to support the development of personalized therapeutic strategies and optimized dosing regimens for animals.
    Keywords:  LC-MS/MS; ampicillin; animals; beta-lactams; liquid chromatography; sulbactam; tandem mass spectrometry; therapeutic dosage
    DOI:  https://doi.org/10.3390/antibiotics14040390
  29. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Apr 26. pii: S1570-0232(25)00179-5. [Epub ahead of print]1259 124625
    Ultra-sensitive quantification of fipronil in zebrafish tissues via UPLC-MS3.
    Meichen Liu, Yingxia Guo, Ziyi Niu, Yufeng Guo, Shuang Feng, Xiaoyan Zhang, Jiansong You, Lei Yin, Hongyu Xue, Meiyun Shi.
      A sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry cubed (UPLC-MS3) assay was established for detection of fipronil in zebrafish tissue samples. Following protein precipitation pretreatment, the concentrations of fipronil in various zebrafish tissue samples were quantified by the developed UPLC-MS3 assay. Fipronil was monitored by MS3 transition (m/z 435.0 → 398.9 → 330.0) with a ± 1.0 Da isolation window for secondary product ions. Chromatographic separation was achieved with 0.1 % formic acid-water (v/v) as the aqueous phase and acetonitrile as the organic phase. Gradient elution with C18 column was employed for separation of fipronil and probenecid (internal standard, IS). Method validation demonstrated excellent linearity over the range of 0.1-10 ng/mL (r2 > 0.995), with the accuracies ranging from -10.33 % to 3.17 % and precisions between 3.25 % and 11.34 %. Consistent extraction recoveries (89.92-113.49 %) and acceptable matrix effects (92.54-110.27 %) were observed across all tested matrices. The implementation of MS3 scanning significantly enhanced specificity by eliminating endogenous interference compared to conventional MRM detection. The successful application of this method in zebrafish tissue distribution studies confirmed its reliability for environmental toxicology research. This MS3-based approach provides a robust analytical platform for investigating biodistribution of fipronil in aquatic models.
    Keywords:  Fipronil; Quantification; Tissue distribution study; UPLC-MS(3); Zebrafish
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124625
  30. Biomolecules. 2025 Apr 17. pii: 594. [Epub ahead of print]15(4):
    Development of an Optimized Two-Step Solid-Phase Extraction Method for Urinary Nucleic Acid Adductomics.
    Alexandra Keidel, Jazmine Virzi, Laura Deloso, Carolina Möller, Dale Chaput, Theresa Evans-Nguyen, Yuan-Jhe Chang, Mu-Rong Chao, Chiung-Wen Hu, Marcus S Cooke.
      The exposome represents the totality of endogenous and exogenous exposures across the lifespan. These exposures may result in DNA and RNA damage, in the form of adducts, which is a key factor in the etiology of a variety of human diseases, including cancer. It is understood that, following their repair, nucleic acid adducts are excreted into the urine, making urine an ideal, non-invasive matrix in which to study the whole-body nucleic acid adductome (the totality of nucleic acid adducts). However, the measurement of these adducts in urine presents challenges due to matrix interference and the variety of the chemical nature across the spectrum of nucleic adducts making their "one-size-fits-all" extraction by solid-phase extraction (SPE) challenging. Here, different types of SPE sorbents, and their combination, were evaluated for maximal recovery of nucleic acid adducts from urine. The SPE column combination of ENV+ coupled with PHE provided the best retention of a cocktail of 20 nucleic acid adduct standards. An untargeted high resolution mass spectrometry approach incorporating FeatureHunter 1.3 software was used to demonstrate the ability of this SPE method to successfully recover endogenous urinary nucleic acid adducts in addition to those represented by the cocktail of isotopically labeled standards. Using our approach, FeatureHunter 1.3 recognized approximately 500 adducts in both mouse and human urine samples. Isotopically labeled standards were used to identify a selection of the endogenous adducts and begin the characterization of the urinary nucleic acid adductome of mice and humans.
    Keywords:  DNA adducts; DNA damage; DNA repair; RNA adducts; adductomics; exposome; mass spectrometry; urine
    DOI:  https://doi.org/10.3390/biom15040594
  31. Rapid Commun Mass Spectrom. 2025 Aug 15. 39(15): e10055
    High-Resolution LC-MS Approach for In Vitro Metabolite Characterisation of Etonitazene in Camels for Enhanced Doping Control.
    Jahfar Nalakath, Ibrahim Waseem Nelliyott, Ahmed M Kadry, Kothandaraman K Sankar, Parseen Ok.
       RATIONALE: The misuse of synthetic opioids particularly those from the benzimidazole class such as etonitazene poses significant challenges for doping control in animal sports including camels racing. These substances are powerful psychoactive agents with a high potential for abuse. Therefore, it is essential to implement robust detection and monitoring strategies to ensure fair competition and protect animal welfare.
    METHODS: In vitro studies were conducted using camel liver homogenates to investigate the metabolism of etonitazene. Metabolites were extracted and analysed using a Thermo Fisher Orbitrap Exploris 120 LC-HRMS system, which provides high-resolution and accurate mass detection. Validation on critical parameters were performed using in-house developed methods while data processing and metabolite characterisation were performed using Compound Discoverer software.
    RESULTS: Six Phase I metabolites of etonitazene have been identified elucidating the metabolic pathways in camels. The metabolism was mainly characterised by dealkylation and nitro reduction processes. These metabolites hold significant potential as biomarkers for the long-term detection of etonitazene for doping control applications in camels.
    CONCLUSION: This study highlights the effectiveness of advanced high-resolution LC-MS techniques in identifying and characterising the in vitro metabolites of etonitazene in camels. Due to its high potency and potential for misuse in camel racing, the identified metabolites serve as a foundation for effective doping control strategies. These findings contribute to improving regulatory frameworks designed to protect animal welfare and maintain the integrity of the sport.
    Keywords:  LC‐HRMS; camel racing; doping control; etonitazene; in vitro metabolism; synthetic opioids
    DOI:  https://doi.org/10.1002/rcm.10055
  32. Res Sq. 2025 Apr 08. pii: rs.3.rs-6321321. [Epub ahead of print]
    Glucuronidation Metabolomic Fingerprinting to Map Host-Microbe Metabolism.
    Andrew Patterson, Nina Boyle, Josh John, Mingxun Wang, Helena Mannochio-Russo, Jeong Joo Pyo, Min Soo Kim, Shuchang Tian, Imhoi Koo, Mallappa Anitha, Yuan Tian, Ethan Morgan, Iain Murray, Gary Perdew, Gary Wu, Pieter Dorrestein, Jordan Bisanz, Matthew Redinbo.
      Glucuronidation is an important detoxification pathway that operates in balance with gastrointestinal microbial β-glucuronidase (GUS) enzymes that can regenerate active metabolites from their glucuronidated forms. Although significant progress has been made in characterizing GUS enzymes, methods to comprehensively define the glucuronidome - the collection of glucuronidated metabolites - remain limited. In this study we employed pattern-filtering data science approaches alongside untargeted LC-MS/MS metabolomics to map the glucuronidome in urine, serum, and colon/fecal samples from gnotobiotic and conventional mice. Our findings reveal microbiome-driven shifts in the glucuronidome, highlighting how differential GUS activity can influence host metabolite profiles. Reverse metabolomics of known glucuronidated chemicals and glucuronidation pattern filtering searches in public metabolomics datasets exposed the diversity of glucuronidated metabolites in human and mouse ecosystems. In summary, we present a new glucuronidation fingerprint resource that provides broader access to and analysis of the glucuronidome. By systematically capturing glucuronidation patterns, this resource enhances unknown metabolite annotation efforts and provides new insights into the dynamic relationship between the host and bacterial biotransformation activities.
    DOI:  https://doi.org/10.21203/rs.3.rs-6321321/v1
  33. Ann Pharm Fr. 2025 Apr 26. pii: S0003-4509(25)00076-8. [Epub ahead of print]
    Unspecified Degradation Impurities Identification and Characterization in Bilastine and Montelukast tablet formulations by using UPLC and LCMS/MS: Robustness by Design Expert and Green assessment.
    Raj Kumar Kodishala, Kousrali Sayyad, Leela Prasad Kowtharapu, Naresh Konduru, Tanmoy Mondal, Mohan Varkolu, Sreedhar Gundekari.
       OBJECTIVES: This study introduces a novel ultraperformance liquid chromatography (UPLC) method for the rapid, simple, and accurate detection of contaminants in bilastine (BLS) and montelukast (MTK) tablet formulations.
    MATERIAL AND METHODS: The separation of BLS impurity-A, BLS impurity-B, BLS, MTK impurity-A, MTK, and MTK impurity-B was achieved using an Acquity BEH C18 column (50 x 2.1 mm, 1.7 μm) under gradient eluent conditions. The mobile phases consisted of 0.1% triethylamine in water with the pH adjusted to 2.5 using orthophosphoric acid (Mobile Phase A), and acetonitrile (Mobile Phase B). The ratio of Mobile Phase A to B was 70:30 (v/v). The column flow rate was set at 0.2 mL/min, and the photodiode array detector (PDA) was used to quantify the analytes. The detection wavelength was set to 224 nm.
    RESULTS: In a runtime of just 20 minutes, 13 analytes were successfully separated. The retention times for the target compounds and impurities were as follows: BLS impurity-A: 1.509 min, BLS impurity-B: 3.435 min, BLS: 5.668 min, MTK impurity-A: 8.137 min, MTK: 9.784 min, MTK impurity-B: 11.853 min. The unspecified impurities in the degradation samples were detected at retention times of 2.174, 2.657, 3.368, 4.143, 8.239, 11.722, and 12.436 minutes, and were characterized using liquid chromatography-mass spectrometry (LC-MS).
    CONCLUSION: The UPLC-based analytical method demonstrated in this study is an effective and efficient technique for the quantification of BLS, MTK, and their associated impurities in tablet formulations. This method has been validated in accordance with ICH Q2(R2) and USP <1225> guidelines.
    Keywords:  Bilastine; Forced degradation; Impurities; Liquid chromatography mass spectrometer; Montelukast; Ultra-performance liquid chromatography
    DOI:  https://doi.org/10.1016/j.pharma.2025.04.006
  34. Anal Chem. 2025 May 02.
    A Facile Workflow for Sebum Fatty Acids and Squalene Identification via Advanced Lipidomic Methodologies.
    Lerong Qi, Xiangzi Li, Yachen Ren, Tingxiang Yang, Yang Guan, Dong Hao Wang, Zhen Wang.
      Sebum fatty acids and squalene are major components of skin secretions and play a critical role in the pathogenesis of dermatological conditions through their compositional alterations. Human sebum fatty acids are uniquely diverse in their chain length, double bond positions, and chain branching; squalene is a rare component of skin secretions with six double bonds. We introduce a facile workflow for the analysis of sebum fatty acids and squalene for elucidating their contribution to the state of skin health or disease using advanced mass spectrometric methods for qualitative and quantitative analyses. Sebum from dermatologically healthy individuals was extracted, and covalent adduct chemical ionization (CACI), electron ionization (EI), and Paternò-Büchi (PB) tandem mass spectrometry (MS/MS) techniques were employed for the identification of fatty acids with varying methyl branch and/or carbon-carbon double bond positions. A method for the full structural characterization of squalene via CACI tandem mass spectrometry was also developed. In sebum, we report for the first time unusual straight-chain polyunsaturated fatty acid isomers (n18:2n-7, n18:2n-3, n20:2n-12, and n20:2n-7), a series of previously unreported branched-chain saturated fatty acids with C19-C26, and a novel branched-chain monounsaturated fatty acid i15:1n-9. A total of 64 fatty acids were identified, exceeding the previous reports. Our study established a comprehensive qualitative and quantitative workflow for the analysis of sebum fatty acids and squalene, with the potential to advance dermatological research and clinical diagnostics.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06010
  35. Anal Chem. 2025 Apr 30.
    Ultrahigh Flow Regulated Discharge Coupling with Targeted Mass Spectrometry Analysis: Real Time Capturing Biomolecules in Exhaled Breath.
    Wenzhao Zhou, Wenting Wang, Mengmeng Jiang, Lanrui Cao, Shichun Shao, Yanna Le, Xudong Fu.
      Online breath analysis is an attractive approach for noninvasive diagnosis of disease and monitoring of human metabolic processes. To date, however, most compounds observed in exhaled breath are volatiles that are not directly associated with blood test results. Here, we proposed an ultrahigh flow regulated discharge (UFD) ionization method that enables online detection of various bioactive breath metabolites and some macromolecules by coupling with mass spectrometry, some of them were commonly observed in blood and body fluids. Through the usage of ultrahigh flow, the corona discharge is transformed into glow discharge in atmospheric pressure, and the mechanism similar to sonic spray may also be involved simultaneously to form a composite ionization manner. Compared to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), UFD exhibited wide coverage and high sample throughput and ionization efficiency for compounds from small volatiles to proteins in different molecular weights (MW). Based on this method, 81 biomolecules can be detected in exhaled breath, including all the 20 common amino acids, nucleosides, tricarboxylic acids, parts of nonprotein amino acids, nucleotides, coenzymes, polyamines, and related derivatives/intermediates. Notably, a total of 63 components were observed for the first time. Moreover, macromolecules also can be detected in exhalation via this method, though these components could not be unambiguously identified due to the limited selectivity. For method validation, the breath homocysteine from five subjects was measured and presented similar trends with blood test results. The online measurement of these biomolecules in human breath showed the possibility of breath tests as an alternative for some blood test items in clinical diagnosis and offers a new approach for real time investigating the metabolic process of nonvolatile bioactive components. Besides, the UFD also provides a new interface for mass spectrometry with a large mass range, wide polarity, high sensitivity, and high throughput.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00195
  36. BMC Vet Res. 2025 Apr 27. 21(1): 294
    Volumetric absorptive microsampling to measure iohexol and creatinine concentrations for estimation of glomerular filtration rate in cats: aligning animal welfare with practical feasibility.
    Hanna De Baets, Marleen Brans, Dominique Paepe, Christophe P Stove.
       BACKGROUND: Chronic kidney disease (CKD) is common in cats, and early detection is crucial for better prognosis. Currently, the gold standard to assess renal function is the measurement of glomerular filtration rate (GFR), allowing early detection of decreased kidney function. To overcome the practical limitations of this procedure, microsampling, collecting a small drop of blood from the cat's ear, can be used. Application of volumetric absorptive microsampling (VAMS) in feline nephrology would be of tremendous value, aligning with animal welfare and improving practical feasibility of GFR measurements.
    RESULTS: We developed and successfully validated liquid chromatography - tandem mass spectrometry (LC-MS/MS) methods to simultaneously determine iohexol and creatinine in plasma, blood and VAMS samples. A clinical validation study, conducted in 23 cats from whom conventional venous blood, plasma and VAMS samples were collected, allowed to establish a conversion formula to derive plasma iohexol or creatinine concentrations from capillary VAMS concentrations. This conversion was applied on an independent set, revealing an excellent agreement for both iohexol and creatinine between concentrations directly measured in venous plasma or derived from ear-prick VAMS samples (94% and 96% of differences lay < 20%, respectively).
    CONCLUSIONS: We demonstrated that ear-prick sampling using VAMS is a suitable alternative to conventional venous sampling to measure iohexol and creatinine for GFR determination in cats.
    Keywords:  Chronic kidney disease; Creatinine; Glomerular filtration rate; Iohexol; Liquid chromatography-tandem mass spectrometry; Volumetric absorptive microsampling
    DOI:  https://doi.org/10.1186/s12917-025-04748-2
  37. Molecules. 2025 Mar 26. pii: 1473. [Epub ahead of print]30(7):
    Dual SPE-HPLC-MS/MS Platform for Cross-Class Antiparasitic Surveillance: Simultaneous Quantification of Oxyclozanide and Levamisole Hydrochloride in Ovine Tissues with Applications to Withdrawal Period Optimization.
    Guonian Dai, Xuzheng Zhou, Weiwei Wang, Bintao Zhai, Jiang Li, Yangling Liu, Yong Zhang, Jiyu Zhang.
      This study presents a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of oxyclozanide (OXY) and levamisole hydrochloride (LEV) residues in ovine tissues, addressing the critical gap in cross-class antiparasitic drug monitoring. Leveraging dual solid-phase extraction strategies-MAX anion-exchange for lipophilic OXY and MCX cation-exchange for hydrophilic LEV-we achieved efficient purification of these pharmacokinetically divergent compounds from complex matrices (muscle, liver, kidney, and perirenal adipose). The method demonstrated superior sensitivity with limits of detection (1.5 μg/kg) and quantification (2.5 μg/kg) below international maximum residue limits (MRLs), validated through Codex Alimentarius guidelines (CAC/GL 71-2009). Linear responses (2.5-1000 μg/kg, R2 > 0.9900) and robust precision (intra-day RSD: 1.44-12.51%; inter-day RSD: 0.29-17.70%) were maintained across spiked concentrations (LOQ, 0.5×, 1×, and 2 × MRLs), with recoveries of 80.94-115.36% confirming matrix-agnostic accuracy. Stability assessments under diverse storage conditions further validated method reliability. Applied to pharmacokinetic profiling in medicated sheep, this protocol established a 28-day withdrawal period for edible tissues, reconciling regulatory compliance with food safety requirements. As the first reported simultaneous quantification platform for OXY and LEV antiparasitics, our methodology advances veterinary residue analytics by enabling efficient multi-class surveillance and evidence-based withdrawal period optimization.
    Keywords:  HPLC–MS/MS; MRLs; oxyclozanide and levamisole hydrochloride suspension; sheep; withdrawal period
    DOI:  https://doi.org/10.3390/molecules30071473
  38. Anal Methods. 2025 May 02.
    Towards the development of an alternative analysis method to determine lead in eyeliner cosmetics.
    Catherine J McMahon, Toni L O Barstis, Rae A Bellows, Charles S Henry.
      Previous research has shown that potentially toxic elements may be present in cosmetic products as impurities or for pigmentation and may be linked to adverse health effects. Yet in low-resource countries where cosmetics hold historical and cultural significance, potentially toxic element contamination in cosmetics is often not well studied or regulated. Current 'gold standard' methods for quantifying these contaminates are inductively coupled plasma - optical emission spectrometry (ICP-OES) and mass spectrometry (ICP-MS). These methods are expensive, time-consuming, and require rigorous sample preparation, making them challenging to perform in low-resource countries. The goal of this research is to develop a field-friendly method of potentially toxic element analysis. Initial studies focused on screening cosmetic samples collected from the low-resource countries; the results showed that lead (Pb) was present in particularly high concentrations. Further analysis showed that 79% of all the collected eyeliner cosmetic samples contained hazardous levels of Pb contamination. Towards the development of a more field-friendly analysis method for Pb contamination, we created and validated a Pb-spiked cosmetic standard that was used to assess different extraction methods. We then optimized an alternative method using citric acid for use with a field-friendly anodic stripping voltammetry analysis method; this method resulted in the detection of 83% of the total Pb present in the fortified standard. However, when this alternative method was used to analyze the collected samples, matrix effects of select cosmetics significantly reduced the Pb detection. Further research will need to be conducted to address the matrix effects of these cosmetics.
    DOI:  https://doi.org/10.1039/d5ay00389j
  39. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 15. pii: S1570-0232(25)00157-6. [Epub ahead of print]1258 124605
    Simultaneous determination of piperacillin, metronidazole, and tazobactam in plasma by UPLC-MS/MS and application to a pharmacokinetic study in pediatric liver transplant patients.
    Cuiyao He, Saisai Ling, Ying Jin, Lisha Fu, Xiaoke Dai, Xiaohui Qi, Yiying Wu, Zhenglei Wang, Yuhua Deng.
      Piperacillin/tazobactam and metronidazole are two commonly antimicrobials used to prevent and treat infections after liver transplantation in pediatric patients. However, under the recommended dose, it may lead to insufficient antimicrobial treatment and poor efficacy. To achieve antimicrobial therapy based on pharmacokinetic strategies, a rapid and highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to measure the plasma concentrations of piperacillin, tazobactam, and metronidazole in pediatric liver transplant recipients. Sample separation was achieved using an Acquity UPLC CSH C18 column (2.1 × 50 mm, 1.7 μm) with gradient elution. The mobile phase consisted of ammonia solution-formic acid-water (0.5:1:1000, v/v/v) and methanol-acetonitrile (1:1, v/v). The plasma concentration range was 0.20-100.00 μg/mL, with good linear range. The intra- and inter-day precisions were less than 13.4 %, and the accuracy ranged from 90.0 % to 109.0 %. The selectivity, carryover, dilution integrity, matrix effect, recovery, and stability met the requirements of the relevant guidelines. The established UPLC-MS/MS method was successfully applied to measure the concentrations of piperacillin, tazobactam, and metronidazole in the plasma of pediatric liver transplant patients. The results showed that according to the recommended dosing regimen, piperacillin/tazobactam reached the effective therapeutic target of 50 %T > MIC in only 4 h. The maximum plasma concentration (Cmax) of metronidazole ranged from 9.46 to 28 μg/mL, with an average Cmax of 16.59 μg/mL, which did not achieve the ideal Cmax/MIC ratio of 8-10.
    Keywords:  Metronidazole; Pediatric liver transplantation; Piperacillin; Tazobactam; UPLC–MS/MS
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124605
  40. Anal Chim Acta. 2025 Jun 22. pii: S0003-2670(25)00427-1. [Epub ahead of print]1356 344033
    Lab-made open-source controlled robotic workstation for sorbent-based dispersive microextraction of low-volume samples.
    Guillem Peris-Pastor, Jose Grau, Luis Arruza, Juan L Benedé, Alberto Chisvert.
       BACKGROUND: Despite microextraction techniques have gained prominence over traditional extraction ones, they usually involve repetitive and tedious manual procedures. Full or partial automation could help alleviate this drawback. Even though commercial systems are available to automate sample preparation, they are very expensive and not affordable for a high number of laboratories.
    RESULTS: With the aim of contributing to automation on sample preparation, and more specifically, on microextraction techniques, a lab-made open-source robotic workstation is presented. It allows to carry out sorbent-based dispersive microextraction in low-volume samples in a semiautomated way. The robotic system is made from low-cost hardware electronic gadgets conveniently assembled in a 3D-printed casing, and it is controlled by the open-source Arduino software. To demonstrate the potential of the developed robotic workstation, it was employed for the determination of three long-chain acylcarnitines in serum of newborns by flow injection analysis-tandem mass spectrometry (FIA-MS/MS). The method was validated in terms of sensitivity, precision and accuracy and it was successfully applied to the analysis of samples from 5 newborns.
    SIGNIFICANCE: The presented robotic workstation is the first non-commercial one dedicated to sorbent-based dispersive microextraction. It is in line with the trends on sample preparation field focused on the development of automated procedures due to their huge advantages over manual procedures. Moreover, the use of open-source and low-cost elements alleviates the high cost of commercially-available systems, which are not affordable for all laboratories.
    Keywords:  Automation; Bioanalysis; Low-volume samples; Microextraction; Open-source technologies; Robotic workstation
    DOI:  https://doi.org/10.1016/j.aca.2025.344033
  41. Biomed Chromatogr. 2025 Jun;39(6): e70064
    Development and Validation of RP-HPLC Method for the Detection of N-Nitroso Meglumine in Pharmaceutical Products.
    Naga Chandrarao Jayavarapu, Shubhalakashmi Sengupta, Hanimi Reddy Bapatu, Praveen Kumar Subbappa.
      Meglumine, a versatile pharmaceutical excipient utilised in the manufacture of numerous pharmaceutical formulations, is an amino sugar containing a secondary amine, which can form a N-nitroso meglumine under nitrosating conditions. These substances are known to be human carcinogens and mutagens. Thus, the development of trustworthy analytical methods has become increasingly essential. This study offers a structural evaluation and development of a reverse phase chromatographic method for the identification and quantification of impurity. The separation was achieved by utilising a Primesep 100, chromatographic column having a mobile phase consisting of water, acetonitrile and formic acid. An HPLC system equipped with photo diode array and refractive index detectors was used. The analytical method was validated in accordance with regulatory standards, demonstrating high specificity with no interference. It exhibited excellent reproducibility, with a % Relative Standard Deviation of 0.56 in system suitability, and a strong linear correlation (0.999) between concentration and response. The method consistently achieved reliable recovery at all spiked levels with a minimum average recovery of 98.0%, maintaining precision at both method concentration and limit of quantification levels, while demonstrating robustness for intended modifications. The successful validation of this method underscores its potential for extensive implementation in pharmaceutical laboratories.
    Keywords:  European Medicines Agency; N‐nitroso meglumine; meglumine; refractive index
    DOI:  https://doi.org/10.1002/bmc.70064
  42. J Mass Spectrom. 2025 May;60(5): e5142
    Development of a Rapid LC-MS/MS Method and Its Application for the Pharmacokinetic Analysis of Pacritinib in Rats.
    Ya Meng Wu, Kai Zheng, Luo Yi Huang, Jing Huan Ni, Peng Fei Tang, Jian Chang Qian, Zhong Xiang Xiao, Yun Lei Li.
      Pacritinib is a novel medication with certain limitations and unknowns in therapeutic applications. Due to the increased frequency of side effects such as fungal infections, nausea, and vomiting, the potential for drug interactions is significantly heightened. This study aimed to establish a quantitative analysis method for pacritinib and investigate its interactions with other drugs. A quantitative detection method for pacritinib in rat plasma was developed using LC-MS/MS, with ibrutinib as an internal standard. This method was subsequently applied to pharmacokinetic and drug-drug interaction studies in rats. The method demonstrated good linearity within the range of 1-1500 ng/mL, with an LLOQ of 1 ng/mL. Both intraday and interday precisions (RSD%) were less than 14.52%, and the recovery, matrix effect, and stability met FDA guidelines. The method proved effective for the quantitative detection of pacritinib in rat plasma. Pharmacokinetic studies revealed that isavuconazole significantly inhibited the metabolism of pacritinib compared to voriconazole, resulting in a 2.5-fold increase in AUC(0-t), a 2.6-fold increase in AUC(0-∞), and a 3.0-fold increase in Cmax. Additionally, the CLz/F value in the isavuconazole group decreased by 67%. This study successfully established a reliable LC-MS/MS method for detecting pacritinib plasma concentrations in rats. The findings indicate that isavuconazole is more likely to increase pacritinib blood exposure than voriconazole, highlighting the need for caution when combining isavuconazole with pacritinib in clinical practice.
    Keywords:  LC‐MS/MS; drug–drug interactions; isavuconazole; pacritinib; voriconazole
    DOI:  https://doi.org/10.1002/jms.5142
  43. Biochem Biophys Res Commun. 2025 Jun 08. pii: S0006-291X(25)00572-8. [Epub ahead of print]765 151858
    Identification of novel oxidized phospholipids that activate platelet-activating factor receptor using HPLC fractionation and comprehensive LC-MS/MS analysis.
    Eisho Kozakura, Ryoya Ueno, Tomohiro Yamashita, Tomomi Hashidate-Yoshida, Hideo Shindou, Mirinthorn Jutanom, Kazushi Morimoto, Ken-Ichi Yamada.
      Platelet-activating factor receptor (PAFR) is involved in various physiological processes, including the immune system and inflammatory responses. In addition to PAF, several oxidized phospholipids have been shown to act as ligands for PAFR. We have previously developed a comprehensive analysis method for oxidized phospholipids, and in this study, we employed this method to test whether additional oxidized phospholipids can activate PAFR. From an oxidized phosphatidylcholine mixture, we identified that 1-palmitoyl-2-(4'-oxo-butanoyl)-sn-glycero-3-phosphocholine (POBPC) functions as a novel PAFR activator, using preparative HPLC and comprehensive LC-MS/MS analysis of fractionated oxidized phospholipids. Next, multiple assays confirmed that POBPC acts as a bona fide PAFR agonist. The H248W mutation of PAFR attenuated the response to POBPC. Finally, POBPC induced phosphorylation of extracellular signal-regulated kinase in mouse peritoneal macrophages, which endogenously express PAFR. Our findings provide valuable insight into the biological functions of oxidized phospholipids, advancing our understanding of their roles in cellular processes.
    Keywords:  HPLC; LC-MS/MS; Oxidized phosphatidylcholine; PAFR; POBPC
    DOI:  https://doi.org/10.1016/j.bbrc.2025.151858
  44. J Pharm Biomed Anal. 2025 Apr 24. pii: S0731-7085(25)00262-6. [Epub ahead of print]263 116921
    Purification of testosterone and its metabolites in urine using two-dimensional high-performance liquid chromatography for 13C/12C ratios analysis by gas chromatography/combustion/isotope ratio mass spectrometry.
    Mengmeng Yan, Tianshuo Zhu, Chao Wen.
      Two-dimensional high-performance liquid chromatography (2D-HPLC) is employed as a sample purification method for the isolation and enrichment of testosterone and its main metabolites in urine samples, thereby facilitating subsequent determination of 13C/12C ratios (δ13C) using Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry (GC/C/IRMS). The orthogonality of the HPLC columns was optimized by leveraging the distinct differences in polarity and steric hindrance effects between the stationary phases, enabling effective separation of endogenous interferents from testosterone in urine. Urine samples purified with 2D-HPLC exhibit significantly reduced matrix interferences in the fractions of testosterone (T), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol), thereby enhancing the accuracy and reliability of GC/C/IRMS analysis. The method achieves limit of quantification as low as 2 ng/mL for testosterone, and 3 ng/mL for both 5β-diol and 5α-diol. Furthermore, the simple instrument configuration enables direct transfer of 1D-purified fractions to 2D-HPLC for further isolation and enrichment, providing a flexible and efficient workflow for method development, particularly in handling complex biological matrices.
    Keywords:  2D-HPLC; Doping control; GC/C/IRMS; Purification; Separation; Testosterone
    DOI:  https://doi.org/10.1016/j.jpba.2025.116921
  45. Nucleic Acids Res. 2025 May 01. pii: gkaf363. [Epub ahead of print]
    MMEASE: enhanced analytical workflow for single-cell metabolomics.
    Qingxia Yang, Yangbo Dai, Shijie Huang, Bing Liu, Huaicheng Sun, Yuan Zhou, Yaguo Gong, Feng Zhu.
      Metabolomics is essential for providing an overview of what chemical processes are taking place. A clear shift from bulk metabolomics to single-cell metabolomics (SCM) is observed in current research, and an integral workflow enabling the analysis of SCM data is therefore in great demand. However, no such workflow has been available to date. Herein, MMEASE, previously designed for analyzing bulk metabolomic data, was therefore updated to its 2.0 version by developing the first comprehensive and in-depth workflow analyzing SCM data. First, it provided all sequential steps of modern SCM research (from SCM data processing, to cellular heterogeneity analysis, then to high-resolution metabolite annotation, and finally to cell-based biological interpretation). Second, compared with the existing tools, MMEASE 2.0 was superior by incorporating the widest variety of methods at every step of the SCM analyses. The originality and functionality of our MMEASE were extensively validated and explicitly described by case studies on benchmark data. All in all, MMEASE 2.0 was unique in accomplishing comprehensive and in-depth analyses of SCM data, which could be considered as an indispensable complement to the existing tools. Now, the latest version of MMEASE is freely accessible by all users at: https://idrblab.org/mmease/.
    DOI:  https://doi.org/10.1093/nar/gkaf363
  46. Talanta. 2025 Apr 08. pii: S0039-9140(25)00602-2. [Epub ahead of print]294 128112
    Development of CE-MS/MS method for unravelling nonsteroidal anti-inflammatory drugs as potential adulterants of Boswellia serrata extract.
    Dmytro Kosolapov, Filip Bařinka, Taťána Gazárková, David Moreno-González, Lucie Nováková, Pavel Jáč.
      The CE-MS/MS method for the analysis of boswellic acids, natural compounds with anti-inflammatory activity, and nonsteroidal anti-inflammatory drugs (NSAIDs) as potential adulterants of dietary supplements with Boswellia serrata extract is presented for the first time. An aqueous-organic background electrolyte comprising 40 mmol/L ammonium acetate (pH 8.5), methanol, and acetonitrile (5:1:4, v/v/v) was used for the separation of boswellic acids and thirteen NSAIDs in negative electrospray ionization mode. Design-of-experiments-guided optimization of Agilent Jet Stream ion source parameters and adjustments to the sheath liquid composition were conducted to improve sensitivity and prevent capillary breakage inside the nebulizer. The CE-MS/MS method was validated for diclofenac, flurbiprofen, ibuprofen, ketoprofen, phenylbutazone, salicylic acid, and carprofen, meeting the ICH M10 guideline requirements for accuracy, precision, matrix effects, and recovery. The lower limits of quantification ranged from 0.04 to 4 μg/mL for all NSAIDs, providing sufficient sensitivity to detect adulteration. The application of the method to dietary supplements revealed no evidence of NSAID adulteration while highlighting quantitative variations in boswellic acid profiles. Further analysis using CE-HRMS with data-dependent acquisition workflow enabled the identification of additional natural triterpenoids and undeclared compounds, such as citric acid and ascorbic acid.
    Keywords:  Adulteration; Boswellia; Boswellic acids; Capillary electrophoresis; Dietary supplements; Mass spectrometry; NSAID
    DOI:  https://doi.org/10.1016/j.talanta.2025.128112
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